Over the past forty years, mechanistic studies of protein folding have focused primarily on proteins fewer than 300 amino acids in length with quite simple folding reactions. Nevertheless, these studies have provided crucial insights into the driving forces and mechanisms of the folding of all proteins, including large proteins with many domains and complex folding reactions. In addition, there is growing recognition that conformational changes coupled to protein functions are mechanistically similar to protein folding reactions. The long term goal of our research is to understand how to use our knowledge based on biophysical studies of simple systems to deduce the folding mechanisms of much more complex proteins and to do so in the context of their function and cellular environment. Specifically, we plan to use conventional kinetic methods such as stopped-flow and temperature-jump fluorescence to measure the folding kinetics of RNase P protein when it binds ligands, particularly its cognate RNA. We also plan to extend our studies on the B domain of protein A (BdpA) to include the A, C, D and E domains so as to understand the global folding this important pathogenicity factor in the bacterium Staphylococcus aureus. Finally, we have developed an experimental approach to study unfolded proteins under physiological conditions and plan to exploit this method to study the unfolded forms of monomeric;repressor and BdpA. We plan to collect a variety of spectroscopic and biophysical data on these model denatured ensembles and compare the ensemble-averaged properties with those of statistical mechanical models of each system. Our goal is to provide a more accurate picture of the significantly populated conformations in these ensembles so that they can be used in our mechanistic models of the folding reaction. PUBLIC HEALTH RELEVANCE: These studies are important because many protein domains have been observed to sample their unfolded states tens or hundreds of times every second, making the unfolded form as relevant to function as the folded form. The biological significance of our proposed studies rests on the relevance of recurrent and complex folding reactions to the regulation and function of proteins in the cell. A detailed understanding of protein-ligand interactions and the myriad biological phenomena that result from them requires a thermodynamic and kinetic description.